When platelets become activated at sites of vascular injury by agonists such as thrombin, collagen, ADP and thromboxane A2, they undergo a reorganization of their actin cytoskeleton. This event is initiated by these agonists activating their cell surface receptors, leading to the formation of lipid second messengers and activation of protein kinases. A great deal has been learned about the critical role of phospholipid second messengers in platelet activation; however, large gaps remain. Based on homology with other phospholipid signaling proteins, we originally proposed that the prominent PKC substrate, pleckstrin, could prove to be a link between G-protein coupled receptors, phospholipid signals and the actin cytoskeleton We have found that over-expressed pleckstrin moderates signals mediated by phospholipase C and phosphatidylinositol 3-kinase, and induces cytoskeletal reorganization through a signaling pathway dependent on integrins and small GTP-binding proteins of the Rho family. It is our hypothesis that pleckstrin moderates phospholipid second messengers formation in platelets, and in concert with integrins, induces actin reorganization contributing to platelet activation. Based on our hypothesis, we propose to do the following: In Specific Aim #1, we will extend our observation that pleckstrin helps regulate the platelet cytoskeleton by determining downstream signaling proteins that are regulated by pleckstrin. Specific Aim #2 will identify the molecules with which pleckstrin interacts in vivo. Specific Aim #3 will examine the consequences of targeted disruption of pleckstrin on signaling on murine megakaryocytes and platelets.